This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-AI-102 Develop diagnostics and drugs for multiple or extensively drug-resistant tuberculosis (MDR/XDR TB). We will develop a rapid and high-throughput method for determining the drug susceptibility profile of tuberculosis patients directly from an acid fast bacilli (AFB) smear. A novel sample processing method and an innovative high throughput DNA sequencing approach will be used to analyze selected genomic regions of Mycobacterium tuberculosis (MTB) for drug-resistance associated mutations. The assay system will be very rapid, sensitive and will operate at very low cost per sample. A key innovative feature is the use of AFB smears as starting material for the drug susceptibility tests. Most cases of tuberculosis in the developing world are diagnosed at microscopy centers. These centers routinely prepare AFB smears, but they lack the capability to perform mycobacterial cultures or molecular tests. Our approach would use existing mail or messenger services to send easily transportable and non-infectious samples to centralized locations for analysis. Centralized diagnostics and surveillance makes sense when ultra high throughput methods and multiplexing permit assays to be performed rapidly and at very low cost. Centralization makes it possible to focus on reducing assay cost and increasing assay speed at a few locations that have the technical expertise to do so. Peripheral clinics can then focus their resources on improving sample transport, and patient outreach rather than developing and maintaining complex assays. Our work will be performed in the following four aims: 1. To create a simple method to extract high-quality MTB DNA from clinical AFB-positive sputum smear slides using bacterial filter capture and bulk acoustic wave mediated cell lysis. 2. To develop an asymmetric barcode PCR and next generation sequencing strategy to test small quantities of extracted MTB DNA for mutations associated with resistance to the first and second line drugs using sample pools and the Roche 454 Titanium sequencing platform. 3. To develop a prototype minifluidic manifold that integrates all steps for simultaneously extracting DNA from 96 AFB slides at one time. 4. To test the system on clinical AFB slides from patients with documented drug susceptible and drug resistant tuberculosis. PUBLIC HEALTH RELEVANCE: We will develop a way to use a small amount of sputum which is smeared on a microscope slide as the starting point for a test of drug resistance in tuberculosis. The test will start when a microscope slide is delivered to the laboratory. Advanced DNA testing technology will then look for mutations within certain tuberculosis genes that can cause drug resistance. Importance to public health: This project will make it possible to choose the correct treatment in patients who have tuberculosis, making them less infectious and increasing the chances for a cure.